運用晶片方式分析蛋白質對軸突生長與前突觸分化之影響

Analyzing protein effects on axons growth and presynaptic differentiation with a chip-based method

許永信(Yung-Hsin Hsu)

指導教授 ： 張兗君

國立清華大學/生命科學院/生命科學系/博士(2012年)

摘要

腦部是一個由眾多神經元網路所構成之多功能且複雜精細的系統；神經元間借由特殊的細胞連結--突觸-進行溝通。每個神經元送出軸突，行進相當距離，然後和適當的目標形成突觸。軸突的生長和突觸的形成，乃是受存於細胞表面，或細胞外環境的眾多蛋白質所調控。為研究此眾多蛋白質對軸突生長和突觸形成的影響。一個以晶片為基礎的系統被開發出來。這一系統含數個微圖案所形成區域。神經元被培養於一區域，而軸突則生長至另遠端另一區域。在這遠端區域，軸突生長及可釋放之突觸囊泡—前突觸形成的標誌，皆可被量化。
在此系統中，可將各種蛋白質塗覆於特定區域上，使軸突暴露於該種蛋白質。藉著測量軸突生長及可釋放之突觸囊泡的數量，用以研究與比較各種蛋白質的效能，。我利用此系統研究neuroligin 1、laminin和collagen對軸突生長及前突觸形成的效能。亦可將單一蛋白質塗覆於不同區域表面，使神經細胞不同部份暴露於該種蛋白質。藉著測量在此條件下前突觸形成及軸突的數量。藉以研究該種蛋白質引發信號的有效傳遞範圍。以此種方法被研究Neuroligin 1顯示其對前突觸形成及軸突成長的信號傳遞，是屬於短距離局部的作用。

關鍵字

軸突生長 ； 前突觸分化

並列摘要

The brain is a versatile system and consists of an intricate network of neurons that communicate with each other through special cell contact sites- synapses. Each neuron sends its axon to travel long distance, to meet appropriate targets and to make synapses. The axonal outgrowth and synaptogenesis are regulated by various proteins which exist on cells surface or in the extracellular environments. For investigating the effects of various exogenous proteins on axonal outgrowth and pre-synaptic specification, a chip-based system has been developed. The system comprises of multiple micropatterned areas mounted on the surface of a glass chip. Neurons are cultured on one area on the chip with their axons being guided to grow in another area on the same surface. The amount of axons and of the clusters of releasing-competent synaptic vesicles (RSV), a marker of pre-synaptic specification, in this latter area can be quantified.
In this system, various proteins can be coated to a specific area where axons grow. By quantifying the different amounts of axons and pre-synaptic specification in this specific area, the effects of different proteins can be compared and investigated. The effects of neuroligin 1, laminin, and collagen on axonal outgrowth as well as pre-synaptic specification have been studied here by using this system. A protein can also be coated at varied areas, and thereby different regions of neurons are exposed to this single protein. The amounts of axons and pre-synaptic specification under such conditions can also be quantified. Thus, the effective signal range of a specific protein that could travel within a neuron can be investigated. By using this method, neurolign1 has been studied and demonstrated to mediate pre-synaptic specification within short ranges.

並列關鍵字

axons growth ； presynaptic differentiation

參考文獻

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